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At least 1,621 records · Page 90Linked to original sources
PubMed1997

Specific in vivo thiol-labeling of the FhuA outer membrane ferrichrome transport protein of Escherichia coli K-12: evidence for a disulfide bridge in the predicted gating loop.

The multifunctional FhuA protein of Escherichia coli K-12 forms a channel that is closed by a loop, tentatively designated the 'gating loop', which is also the principal binding site for all FhuA ligands. In this report, it is shown by in vivo labeling that the two cysteines in the gating loop form a disulfide bridge, and they react weakly after reduction with biotin-maleimide, as determined by streptavidin-beta-galactosidase bound to biotin. The two cysteines close to the C-terminus of FhuA also form a disulfide bridge and react with the thiol reagents only after heat denaturation of FhuA in SDS. Replacement of the existing cysteines by serine did not alter the sensitivity of cells to the FhuA ligands tested (T5, phi 80, T1, colicin M, and albomycin) and supported growth on ferrichrome as sole iron source. The cysteines in the gating loop play no specific functional role; they are largely buried in the interior of the loop, and the disulfide bridges are not essential for maintaining the conformation of FhuA. The C-terminal cysteines are in the interior of FhuA and are also not important for the structure of FhuA. The method used allows the identification of free cysteines and disulfides in surface exposed protein regions.

Amino Acid Sequence↗
PubMed2006

Characterization of novel M-superfamily conotoxins with new disulfide linkage.

The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

Amino Acid Sequence↗
PubMed2006

Aberrant interchain disulfide bridge of tissue-nonspecific alkaline phosphatase with an Arg433-->Cys substitution associated with severe hypophosphatasia.

Various mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia characterized by defective bone and tooth mineralization; however, the underlying molecular mechanisms remain largely to be elucidated. Substitution of an arginine at position 433 with a histidine [TNSALP(R433H)] or a cysteine [TNSALP(R433C)] was reported in patients diagnosed with the mild or severe form of hypophosphatasia, respectively. To define the molecular phenotype of the two TNSALP mutants, we sought to examine them in transient (COS-1) and conditional (CHO-K1 Tet-On) heterologous expression systems. In contrast to an 80 kDa mature form of the wild-type and TNSALP(R433H), a unique disulfide-bonded 160 kDa molecular species appeared on the cell surface of the cells expressing TNSALP(R433C). Sucrose density gradient centrifugation demonstrated that TNSALP(R433C) forms a disulfide-bonded dimer, instead of being noncovalently assembled like the wild-type. Of the five cysteine residues per subunit of the wild-type, only Cys102 is thought to be present in a free form. Replacement of Cys102 with serine did not affect the dimerization state of TNSALP(R433C), implying that TNSALP(R433C) forms a disulfide bridge between the cysteine residues at position 433 on each subunit. Although the cross-linking did not significantly interfere with the intracellular transport and cell surface expression of TNSALP(R433C), it strongly inhibited its alkaline phosphatase activity. This is in contrast to TNSALP(R433H), which shows enzyme activity comparable to that of the wild-type. Importantly, addition of dithiothreitol to the culture medium was found to partially reduce the amount of the cross-linked form in the cells expressing TNSALP(R433C), concomitantly with a significant increase in enzyme activity, suggesting that the cross-link between two subunits distorts the overall structure of the enzyme such that it no longer efficiently carries out its catalytic function. Increased susceptibility to proteases confirmed a gross conformational change of TNSALP(R433C) compared with the wild-type. Thus, loss of function resulting from the interchain disulfide bridge is the molecular basis for the lethal hypophosphatasia associated with TNSALP(R433C).

Alkaline Phosphatase↗
PubMed2004

Disulfide-dependent multimeric assembly of resistin family hormones.

Resistin, founding member of the resistin-like molecule (RELM) hormone family, is secreted selectively from adipocytes and induces liver-specific antagonism of insulin action, thus providing a potential molecular link between obesity and diabetes. Crystal structures of resistin and RELMbeta reveal an unusual multimeric structure. Each protomer comprises a carboxy-terminal disulfide-rich beta-sandwich "head" domain and an amino-terminal alpha-helical "tail" segment. The alpha-helical segments associate to form three-stranded coiled coils, and surface-exposed interchain disulfide linkages mediate the formation of tail-to-tail hexamers. Analysis of serum samples shows that resistin circulates in two distinct assembly states, likely corresponding to hexamers and trimers. Infusion of a resistin mutant, lacking the intertrimer disulfide bonds, in pancreatic-insulin clamp studies reveals substantially more potent effects on hepatic insulin sensitivity than those observed with wild-type resistin. This result suggests that processing of the intertrimer disulfide bonds may reflect an obligatory step toward activation.

Adipocytes↗
PubMed2000

Redox signaling in chloroplasts: cleavage of disulfides by an iron-sulfur cluster.

Light generates reducing equivalents in chloroplasts that are used not only for carbon reduction, but also for the regulation of the activity of chloroplast enzymes by reduction of regulatory disulfides via the ferredoxin:thioredoxin reductase (FTR) system. FTR, the key electron/thiol transducer enzyme in this pathway, is unique in that it can reduce disulfides by an iron-sulfur cluster, a property that is explained by the tight contact of its active-site disulfide and the iron-sulfur center. The thin, flat FTR molecule makes the two-electron reduction possible by forming on one side a mixed disulfide with thioredoxin and by providing on the opposite side access to ferredoxin for delivering electrons.

Amino Acid Motifs↗
PubMed2006

Roles of the intramolecular disulfide bridge in MotX and MotY, the specific proteins for sodium-driven motors in Vibrio spp.

The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na+-driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented the formation of an intramolecular disulfide bond, which is presumably important for protein stability. Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge for proper function.

Amino Acid Sequence↗
PubMed1983

Role of primary structure and disulfide bond formation in beta-lactamase secretion.

Plasmid pBR322-encoded beta-lactamase was shown to contain a single disulfide bond, which caused the protein to migrate faster in sodium dodecyl sulfate-polyacrylamide gels than the fully reduced form. A similar difference in mobility of the in vitro synthesized precursor before and after reduction indicates that it also contained a disulfide bond. Formation of the disulfide bond in vivo, however, occurred concomitant with processing. In vivo accumulation of the precursor by inhibition of secretion did not allow disulfide bond formation to occur. This result is consistent with post-translational translocation of the precursor. Synthesis of a fragment of beta-lactamase lacking the carboxy terminus was obtained by insertion of a foreign DNA segment into the PstI site of bla. Processing and secretion of the protein did not appear to be greatly affected, indicating that the carboxy terminus is not required for secretion.

Amino Acid Sequence↗
PubMed1995

Cloning and characterization of the gene for a protein thiol-disulfide oxidoreductase in Bacillus brevis.

The gene (bdb) for protein thiol-disulfide oxidoreductase cloned from Bacillus brevis was found to encode a polypeptide consisting of 117 amino acid residues with a signal peptide of 27 residues. Bdb contains a well-conserved motif, Cys-X-X-Cys, which functions as the active center of disulfide oxidoreductases such as DsbA, protein disulfide isomerase, and thioredoxin. The deduced amino acid sequence showed significant homology with those of several bacterial thioredoxins. The bdb gene complemented the Escherichia coli dsbA mutation, restoring motility by means of flagellar and alkaline phosphatase activity. The Bdb protein overproduced in B. brevis was enzymatically active in both reduction and oxidization of disulfide bonds in vitro. Immunoblotting indicated that Bdb could function at the periphery of the cell.

Amino Acid Sequence↗
PubMed1998

Contribution of the disulfide bond of the A subunit to the action of Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (LT) consists of an A subunit and five B subunits. These subunits oligomerize into an assembled holotoxin within the periplasm. Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus. This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm. However, it is not known whether other regions of the A subunit contribute to the assembly. The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199. It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein. We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit. We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes. The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly. A reconstitution experiment in vitro supported the notion. Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs. These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.

Amino Acid Sequence↗
PubMed2000

A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum.

Several lactic acid bacteria produce so-called pediocin-like bacteriocins that share sequence characteristics, but differ in activity and target cell specificity. The significance of a C-terminal disulfide bridge present in only a few of these bacteriocins was studied by site-directed mutagenesis of pediocin PA-1 (which naturally contains the bridge) and sakacin P (which lacks the bridge). Introduction of the C-terminal bridge into sakacin P broadened the target cell specificity of this bacteriocin, as illustrated by the fact that the mutants were 10 to 20 times more potent than the wild-type toward certain indicator strains, whereas the potency toward other indicator strains remained essentially unchanged. Like pediocin PA-1, disulfide-containing sakacin P mutants had the same potency at 20 and 37 degrees C, whereas wild-type sakacin P was approximately 10 times less potent at 37 degrees C than at 20 degrees C. Reciprocal effects on target cell specificity and the temperature dependence of potency were observed upon studying the effect of removing the C-terminal disulfide bridge from pediocin PA-1 by Cys-->Ser mutations. These results clearly show that a C-terminal disulfide bridge in pediocin-like bacteriocins contributes to widening of the antimicrobial spectrum as well as to higher potency at elevated temperatures. Interestingly, the differences between sakacin P and pediocin PA-1 in terms of the temperature dependency of their activities correlated well with the optimal temperatures for bacteriocin production and growth of the bacteriocin-producing strain.

Amino Acid Sequence↗
PubMed2006

The structure of G4, the poxvirus disulfide oxidoreductase essential for virus maturation and infectivity.

The possibility of the release of smallpox virus into a predominantly nonimmunized population highlights the importance of understanding poxvirus biology. Poxviruses encode a conserved pathway that is required to oxidize disulfide bonds in nascent viral proteins that fold in the reducing environment of the eukaryotic host cytoplasm. We present the structure of the last enzyme of the vaccinia virus pathway, G4, which is almost identical in smallpox virus. G4 catalyzes the formation of disulfide bonds in proteins that are critical for virus maturation and host cell infection. G4 contains a thioredoxin fold and a Cys-X-X-Cys active site. In solution, G4 monomers and dimers are observed. In the crystal, G4 is found as a dimer that buries 4,500 A(2) in the interface and occludes the active site, which could protect the reactive disulfide from reduction in the cytoplasm. The structure serves as a model for drug design targeting viral disulfide bond formation.

Amino Acid Sequence↗
PubMed1980

Protein-protein interactions within paramyxoviruses identified by native disulfide bonding or reversible chemical cross-linking.

Analysis of native disulfide-bonded protein oligomers in paramyxoviruses showed that some viral proteins are consistently present as covalent complexes. In isolated Sendai virus the hemagglutinating protein HN is present in homodimeric and homotetrameric forms, and the minor nucleocapsid protein P exists partly as a monomer and partly as a disulfide-linked homotrimer. Similar disulfide-linked complexes were observed in Newcastle disease virus (strain HP-16), in which HN exists as a homodimer and some of the major nucleocapsid protein NP exists as a homotrimer. Noncovalent intermolecular interactions between proteins were studied with the reversible chemical cross-linkers dimethyl-3,3'-dithiobispropionimidate and methyl 3-[(p-azidophenyl)dithio]propionimidate, which contain disulfide bridges and a 1.1-nm separation between their functional groups. The same results were achieved with both reagents. The conditions of preparation, isolation, and storage of the viruses affected the protein-protein interactions observed upon cross-linking. Homooligomers of the glycoprotein F, the matrix protein M, and the major nucleocapsid protein NP were produced in both Sendai and Newcastle disease viruses after mild cross-linking of all viral preparations examined, but NP-M heterodimer formation in both viruses was most prevalent in early harvest preparations that were cross-linked soon after isolation. The ability of NP and M to form a heterodimer upon cross-linking indicates that the matrix protein layer lies in close proximity (within 1.1 nm) to the nucleocapsid in the newly formed virion. Some noncovalent intermolecular protein interactions in Sendai and Newcastle disease viruses, i.e., those leading to the formation of F, NP, and M homooliogmers upon cross-linking, are more stable to virus storage than others, i.e., those leading to the formation of an NP-M heterodimer upon cross-linking. The storage-induced loss of the ability of NP and M to form a heterodimer is not accompanied by any apparent loss of infectivity. This indicates that some spacial relationships which form during virus assembly can alter after particle formation and are not essential for the ensuing stages of the infectious process.

Azides↗
PubMed1981

Identification of the P proteins and other disulfide-linked and phosphorylated proteins of Newcastle disease virus.

A unique abundant protein, designated P by analogy to the putative polymerase proteins of other paramyxoviruses, was identified in purified Newcastle disease virus. Under nonreducing conditions the P proteins could be separated from other viral proteins on sodium dodecyl sulfate-polyacrylamide gels. The P proteins were isolated from detergent-solubilized virions as 53,000- to 55,000-dalton monomers and disulfide-linked trimers. Distinct forms of P having four different isoelectric points and two different electrophoretic mobilities were resolved by two-dimensional electrophoresis. Two forms of P were phosphorylated, as were the nucleocapsid protein and non-glycosylated membrane protein. In addition to disulfide-linked forms of P, dimers of the hemagglutinin-neuraminidase glycoprotein and two disulfide-linked versions of the fusion glycoprotein were identified. Several electrophoretic variants of the nucleocapsid protein that were probably created by intrachain disulfide bonding were also isolated from virions under nonreducing conditions. The locations of the newly identified proteins were determined by detergent-salt fractionation of virions and by surface-selective radioiodination of the viral envelope. The P proteins were associated with nucleocapsids and were not detected at the surface of virions. Both forms of the fusion glycoproteins were on the exterior of the viral envelope. Herein the properties of the P proteins are compared with similar proteins of rhabdoviruses and other paramyxoviruses, and a role for multiple forms of proteins in the genetic economy of newcastle disease virus is discussed.

Disulfides↗
PubMed1989

Leukemogenic membrane glycoprotein encoded by Friend spleen focus-forming virus: transport to cell surfaces and shedding are controlled by disulfide-bonded dimerization and by cleavage of a hydrophobic membrane anchor.

The leukemogenic glycoprotein (gp55) encoded by Friend spleen focus-forming virus is predominantly retained in the rough endoplasmic reticulum (RER). However, a small proportion (ca. 5%) is processed to form a derivative that occurs on plasma membranes and causes mitosis of infected erythroblasts. We have now found that gp55 folds heterogeneously in the RER to form components with different disulfide bonds and that this difference may determine their processing fates. RER gp55 consists predominantly of monomers with intrachain disulfide bonds. In contrast, the processed molecules are disulfide-bonded dimers. These dimers are extensively modified in transit to cell surfaces by conversion of four N-linked high-mannose oligosaccharides to complex derivatives and by attachment of a sialylated O-linked oligosaccharide. The plasma membrane dimers are then slowly shed into the medium by a mechanism that involves proteolytic cleavage of approximately 25 membrane-anchoring hydrophobic amino acids from the carboxyl termini of the glycoproteins. Consequently, shed molecules have shorter polypeptide chains than cell-associated gp55. We conclude that gp55 folds into different disulfide-bonded components that do not substantially isomerize, and that only one specific dimer is competent for export from the RER. Mitogenic activity of gp55 could be caused by the cell surface dimers, by the shed derivative, or by the carboxyl-terminal hydrophobic anchors that remain in the membranes after the shedding reaction.

Biological Transport↗
PubMed1991

Differential formation of disulfide linkages in the core antigen of extracellular and intracellular hepatitis B virus core particles.

Our understanding of the assembly of hepatitis B virus is still very limited. We present evidence to demonstrate that the HBc antigen formed oligomers through disulfide linkages in the extracellular hepatitis B virus core (HBc) particles. However, the intracellular HBV core particles did not contain disulfide-linked HBc antigens. Furthermore, the extracellular particles which had disulfide bonds were more stable than intracellular particles at pH 7.5 and 10 and in 3 M NaCl and 4 M urea. These data suggest that the formation of disulfide bonds in the HBc antigen is important for the integrity of the viral core particles.

Capsid↗
PubMed1992

Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin.

At a low pH, the influenza virus hemagglutinin (HA) undergoes conformational changes that promote membrane fusion. While the critical role of fusion peptide release from the trimer interface has been demonstrated previously, the role of globular head dissociation in the overall fusion mechanism remains unclear. To investigate this question, we have analyzed in detail the fusion activity and low pH-induced conformational changes of a mutant, Cys-HA, in which the globular head domains are locked together by engineered intermonomer disulfide bonds (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufmann, E. Suchanek, C. Pabo, J. J. Skehel, D. C. Wiley, and S. Wharton, Cell 68:635-645, 1992). In this paper, we show that Cys-HA expressed on the cell surface is predominantly a disulfide-bonded trimer. Cell surface Cys-HA is impaired in its membrane fusion activity, as demonstrated by both content-mixing and lipid-mixing fusion assays. It is also impaired in its ability to change conformation at a low pH, as assessed by proteinase K sensitivity. The fusion activity and low pH-induced conformational changes of cell surface Cys-HA are, however, restored to nearly wild-type levels upon reduction of the intermonomer disulfide bonds. By using a set of conformation-specific monoclonal and anti-peptide antibodies, we found that purified Cys-HA trimers are impaired in changes that occur in the globular head domain interface. In addition, changes that occur at a great distance from the engineered intermonomer disulfide bonds, notably release of the fusion peptides, are also impaired. Our results are discussed with respect to current views of the fusion-active conformation of the HA trimer.

Animals↗
PubMed1993

Disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins.

We have analyzed the effects of reducing conditions on the folding of the spike (S) protein and on the intracellular transport of the membrane (M) protein of the mouse hepatitis coronavirus. These proteins differ in their potential to form disulfide bonds in the lumen of the endoplasmic reticulum (ER). Intrachain disulfide bonds are formed in the S protein but not in M, which was demonstrated in a pulse-chase experiment by analyzing the viral proteins under nonreducing conditions. To reduce disulfide bonds in vivo, we added dithiothreitol (DTT) to the culture medium of mouse hepatitis coronavirus-infected cells following a procedure recently described by Braakman et al. (I. Braakman, J. Helenius, and A. Helenius, EMBO J. 11:1717-1722, 1992). Short exposure to DTT resulted in the complete reduction of newly synthesized S protein and affected its conformation as judged by the change in mobility in nonreducing gels and by the loss of recognition by a conformation-specific monoclonal antibody. Using this antibody in an immunofluorescence assay, we monitored the reducing effect of DTT in situ. DTT was found to initially affect only the S protein present in the ER; also, after longer treatment, the remaining signal also gradually disappeared. In contrast, folding and transport of the M protein were not inhibited by DTT. Under reducing conditions, M was transported efficiently to the trans side of the Golgi complex, indicating that cellular processes such as ER-to-Golgi transport, O-glycosylation, and Golgi retention were unaffected. In the presence of DTT, the M protein even moved at an increased rate to the Golgi complex, which is probably because of its failure to interact with unfolded S protein. The effects of in vivo reduction were reversible. When DTT was removed from pulse-labeled cells, the S protein folded posttranslationally and aberrantly; during its oxidation, most of S now transiently aggregated into large disulfide-linked complexes from which subsequently folded S molecules dissociated.

Biological Transport↗
PubMed1994

Assignment of disulfide bridges in the fusion glycoprotein of Sendai virus.

The mature fusion (F) glycoprotein of the paramyxovirus family consists of two disulfide-linked subunits, the N-terminal F2 and the C-terminal F1 subunits, and contains 10 cysteine residues which are highly conserved at specific positions. The high level of conservation strongly suggests that they are indeed disulfide linked and play important roles in the folding and functioning of the molecule. However, it has not even been clarified which cysteine residues link the F2 and F1 subunits. This report describes our assignment of the disulfide bridges in purified Sendai virus F glycoprotein by fragmentation of the polypeptide and isolation of cystine-containing peptides and determination of their N-terminal sequences. The data demonstrate that all of the 10 cysteine residues participate in disulfide bridges and that Cys-70, the only cysteine in F2, and Cys-199, the most upstream cysteine in F1, form the interchain bond. Of the remaining eight cysteine residues clustered near the transmembrane domain of F1, the specific bridges identified are Cys-338 to Cys-347 and Cys-362 to Cys-370. Although no exact pairings between the subsequent four residues were defined, it seems likely that the most downstream, Cys-424, is linked to Cys-394, Cys-399, or Cys-401. Thus, we conclude that the cysteine-rich domain indeed contributes to the formation of a bunched structure containing at least two tandem cystine loops.

Amino Acid Sequence↗